Antibody-dependent cellular cytotoxicity (ADCC) contributes to clinical efficacy of some unconjugated monoclonal antibodies. The overall goal of this research proposal is to employ the techniques of antibody engineering to optimize ADCC and then determine the mechanism(s) by which ADCC promotes anti-tumor effects. We will address two broad hypotheses: 1) Antibodies with high affinity both for a tumor antigen and for cellular Fc receptors will exhibit highly efficient ADCC and will mediate potent in vivo anti-tumor effects;and 2) Such antibodies will induce adaptive immune responses that contribute to tumor efficacy. To test these hypotheses, the C6.5 series of anti-human HER2/neu single-chain Fv molecules (scFv) has been converted into fully human lgG1 molecules that identify an identical epitope on the HER2/neu extracellular domain (ECD) with intrinsic binding site affinities ranging from 10(-7) to 10(-11) M. Preliminary data indicate that the capacity of these antibodies to promote in vitro ADCC is related to their intrinsic binding site affinities. These antibodies provide a unique resource to test the role of intrinsic binding site affinity on ADCC and in vivo efficacy. We have created an immunocompetent mouse strain that is transgenic for human HER2/neu and thus permits study of the link between ADCC and the induction of adaptive immune responses. The first specific aim is to prepare and characterize engineered human lgG1 monoclonal antibodies that bind to human HER2/neu and to human Fc-gammaRIII with varying affinities. The second specific aim is to define the contribution of innate immune system mechanisms to anti-tumor efficacy by assessing the in vivo anti-tumor effects of antibodies with varying affinity properties in immunodeficient SCID mice bearing human tumor xenografts that overexpress human HER2/neu. In the third specific aim we will determine the ability of selected efficacious antibodies to induce host-protective adaptive immune responses against human HER2/neu and to tumors expressing that antigen. These studies also will test the hypothesis that treatment-induced in vivo ADCC-promoted antigen presentation initiates an adaptive immune response that contributes to tumor control and can be further amplified and prolonged by promoting T cell costimulation to expand antigen-specific cytotoxic T-cells. The studies proposed here will lead to the creation of antibodies that mediate maximal ADCC and promote the induction of immune responses directed against autologous tumors in immunocompetent subjects. Strategies to amplify and sustain that anti-tumor immune response will be identified. A candidate antibody for clinical development will emerge, as will generally useful information regarding the structural features of antibodies that contribute to ADCC and in vivo efficacy.